Smear-positive Specimens Research Articles

Evaluation of the Speed-oligo Direct Mycobacterium tuberculosis Assay for Molecular Detection of Mycobacteria in Clinical Respiratory Specimens

We present the first evaluation of a novel molecular assay, the Speed-oligo Direct Mycobacterium tuberculosis (SO-DMT) assay, which is based on PCR combined with a dipstick for the detection of mycobacteria and the specific identification of M. tuberculosis complex (MTC) in respiratory specimens. A blind evaluation was carried out in two stages: first, under experimental conditions on convenience samples comprising 20 negative specimens, 44 smear- and culture-positive respiratory specimens, and 11 sputa inoculated with various mycobacterium-related organisms; and second, in the routine workflow of 566 fresh respiratory specimens (4.9% acid-fast bacillus [AFB] smear positives, 7.6% MTC positives, and 1.8% nontuberculous mycobacteria [NTM] culture positives) from two Mycobacterium laboratories. SO-DMT assay showed no reactivity in any of the mycobacterium-free specimens or in those with mycobacterium-related organisms. Compared to culture, the sensitivity in the selected smear-positive specimens was 0.91 (0.92 for MTC and 0.90 for NTM), and there was no molecular detection of NTM in a tuberculosis case or vice versa. With respect to culture and clinical data, the sensitivity, specificity, and positive and negative predictive values for the SO-DMT system in routine specimens were 0.76 (0.93 in smear positives [1.0 for MTC and 0.5 for NTM] and 0.56 in smear negatives [0.68 for MTC and 0.16 for NTM]), 0.99, 0.85 (1.00 in smear positives and 0.68 in smear negatives), and 0.97, respectively. Molecular misidentification of NTM cases occurred when testing 2 gastric aspirates from two children with clinically but not microbiologically confirmed lung tuberculosis. The SO-DMT assay appears to be a fast and easy alternative for detecting mycobacteria and differentiating MTC from NTM in smear-positive respiratory specimens.

GenoType MTBDRsl Line Probe Assay Shortens Time to Diagnosis of Extensively Drug-Resistant Tuberculosis in a High-Throughput Diagnostic Laboratory

Conventional culture-based drug susceptibility testing (DST) for the second-line antituberculosis drugs is slow, leading to diagnostic delay with associated exacerbation of transmission, amplification of resistance, and increased mortality. To assess the diagnostic performance of the GenoType MTBDRsl line probe assay (LPA) for the rapid detection of mutations conferring resistance to ofloxacin (OFX), amikacin (AMK), and ethambutol and to determine the impact of implementation on the turnaround time in a high-throughput diagnostic laboratory. Six hundred and fifty-seven direct patient acid-fast bacilli smear-positive specimens resistant to isoniazid, rifampin, or both according to the GenoType MTBDRplus assay were consecutively tested, using the GenoType MTBDRsl LPA. The diagnostic performance was assessed relative to the "gold standard" culture-based method, and the laboratory turnaround times for both methods were determined. A total of 516 of 657 patient specimens had valid results for both tests. The sensitivity for detecting OFX, AMK, and extensive drug resistance, using the GenoType MTBDRsl LPA, was 90.7% (95% confidence interval [CI], 80.1-96.0%), 100% (95% CI, 91.8-100%), and 92.3% (95% CI, 75.9-97.9%), respectively, and the specificity for detection was 98.1% (95% CI, 96.3-99.0%), 99.4% (95% CI, 98.2-99.8%), and 99.6% (95% CI, 98.5-99.9%), respectively. Implementation of this test significantly reduced the turnaround time by 93.3% (P < 0.001), calculated from the date that the specimen was received at the laboratory to reporting second-line results. In addition, a significant increase in diagnostic yield of 20.1% and 19.3% (P < 0.001) for OFX and AMK resistance, respectively, was obtained for isolates that were either contaminated or had lost viability. The GenoType MTBDRsl LPA is a rapid and reliable DST that can be easily incorporated into the diagnostic algorithm. This assay significantly improved diagnostic yield (P < 0.001) while simultaneously decreasing diagnostic delay for reporting second-line DST. The rapid dissemination of second-line DST results will guide initiation of appropriate treatment, thereby reducing transmission and improving treatment outcome.

The Diagnostic Performance of the GenoType MTBDR plus Version 2 Line Probe Assay Is Equivalent to That of the Xpert MTB/RIF Assay

Molecular diagnostics for Mycobacterium tuberculosis have recently been endorsed by the World Health Organization. The Xpert MTB/RIF assay was endorsed for use on patient material, regardless of smear gradation, while the GenoType MTBDRplus (version 1) has been limited for use on smear-positive patient material. In this study, we evaluated the diagnostic performance of the Xpert MTB/RIF and GenoType MTBDRplus (version 2) assays on smear-positive and smear-negative patient specimens submitted to a high-throughput diagnostic laboratory. A total of 282 consecutive specimens were subjected to the two new molecular assays, and their performance characteristics were assessed relative to the routine diagnostic standard. Both assays showed similar diagnostic performance characteristics. The sensitivities of the GenoType MTBDRplus (v2.0) and Xpert MTB/RIF assays for the detection of culture-positive M. tuberculosis were 73.1% and 71.2%, respectively, while the specificities of both assays were 100%. Both assays were able to diagnose the presence of M. tuberculosis in 57 to 58% of smear-negative cases, suggesting that the performance characteristics were dependent on bacillary load. The detection of M. tuberculosis in culture-negative specimens confirmed that molecular assays should not be used for treatment monitoring. The sensitivity and specificity for rifampin resistance detection were 100% in both assays; however, the GenoType MTBDRplus (v2.0) assay provided additional information on isoniazid susceptibility. The GenoType MTBDRplus (v2.0) assay will complement the Xpert MTB/RIF screening assay by validating rifampin susceptibility and providing information on isoniazid susceptibility. In addition, the GenoType MTBDRplus (v2.0) assay will provide pharmacogenetic information that may be critical in guiding appropriate treatment.

Comparison of two in-house real-time PCR assays with MTB Q-PCR Alert and GenoType MTBDRplus for the rapid detection of mycobacteria in clinical specimens

An in-house IS6110 real-time PCR (IH IS6110), MTB Q-PCR Alert (Q-PCR) and GenoType MTBDRplus (MTBDR; Hain Lifescience) were compared for the direct detection of Mycobacterium tuberculosis complex (MTBC) in 87 specimens following automated NucliSENS easyMAG DNA extraction. This included 82 first smear-positive specimens and three smear-negative specimens. Another in-house real-time PCR with a Mycobacterium genus-specific probe for the internal transcribed spacer (ITS) region (IH ITS) was used to allow a full comparison with culture results. The sensitivities of IH IS6110, Q-PCR, MTBDR and IH ITS for MTBC detection were 100, 92, 87 and 87 %, respectively, compared with culture. Both IS6110-based real-time PCRs (in-house and Q-PCR) were similar in performance, with 91.2 % concordant results for MTBC detection. Inhibition rates were low, with zero to three specimens producing uninterpretable results. However, the Q-PCR failed to detect MTBC in five samples that were smear negative or had few acid-fast bacilli (one to 10 bacilli in 10 microscopic fields) detected by IH IS6110. IH ITS was the least sensitive assay but may be useful when used in conjunction with IS6110 PCR results to determine the presence of non-tuberculous mycobacteria in smear-negative specimens. None of the real-time PCR assays tested provided drug-resistance data. It was concluded that an IH IS6110 assay could easily be incorporated into the workflow of a diagnostic laboratory for rapid and accurate identification of MTBC from clinical specimens. The inclusion of an internal control and amplification of an ITS target enhance the diagnostic utility of the test.

Oral Antimicrobial Rinse to Reduce Mycobacterial Culture Contamination among Tuberculosis Suspects in Uganda: A Prospective Study

RationaleContamination by bacterial or fungal organisms reduces the effectiveness of mycobacterial culture for diagnosis of pulmonary tuberculosis (TB). We evaluated the effect of an anti-microbial and an anti-fungal oral rinse prior to expectoration on culture-contamination rates.MethodsWe enrolled a consecutive random sample of adults with cough for ≥2 weeks and suspected TB admitted to Mulago Hospital (Kampala, Uganda) between October 2008 and June 2009. We randomly assigned patients to oral rinse (60 seconds with chlorhexidine followed by 60 seconds with nystatin) vs. no oral rinse prior to initial sputum collection. Uganda National Tuberculosis Reference Laboratory technicians blinded to the method of sputum collection (with or without oral rinse) processed all sputum specimens for smear microscopy (direct Ziehl-Neelsen) and mycobacterial culture (Lowenstein-Jensen media).ResultsOf 220 patients enrolled, 177 (80%) were HIV-seropositive (median CD4-count 37 cells/uL, IQR 13–171 cells/uL). Baseline characteristics were similar between patients in the oral-rinse (N = 110) and no oral-rinse (N = 110) groups. The proportion of contaminated cultures was significantly lower in the oral-rinse group compared to the no oral-rinse group (4% vs. 15%, risk difference −11%, 95% CI −18 to −3%, p = 0.005). Oral rinse significantly reduced the proportion of contaminated cultures among HIV-infected patients (3% vs. 18%, risk difference −14%, 95% CI −23 to −6%, p = 0.002) but not HIV-uninfected (6% vs. 4%, risk difference 2%, 95% CI −12 to +15%, p = 0.81) patients. However, the proportion of smear-positive specimens (25% vs. 35%, p = 0.10) and culture-positive specimens (48% vs. 56%, p = 0.24) were lower in the oral-rinse compared to the no oral-rinse group, although the differences were not statistically significant.ConclusionsOral rinse prior to sputum expectoration is a promising strategy to reduce mycobacterial culture contamination in areas with high HIV prevalence, if strategies can be devised to reduce the adverse impact of oral rinse on smear- and culture-positivity.

Diagnosis of tuberculosis and drug resistance: what can new tools bring us? [State of the art series. New tools. Number 1 in the series

This is an exciting time for tuberculosis (TB) diagnostics. The technology for rapid diagnosis of TB and rifampicin (RMP) resistance in pulmonary sputum smear-positive specimens is well advanced, and assays have high specificity with good sensitivity. Nevertheless, the current sensitivity of TB detection means that these assays still cannot replace the standard diagnostic methods for TB or conventional drug susceptibility testing (DST). In extra-pulmonary specimens, the performance of molecular tools varies and should be considered separately for each specimen type. Evidence for the use of these assays for TB and drug resistance detection in individuals co-infected with TB and the human immunodeficiency virus (HIV) is limited. As the positive predictive value for RMP resistance reaches ≥ 90% only when the prevalence of RMP resistance in new TB patients is >15%, which is rare globally, many cases with such resistance will be false-resistant, emphasising the need for a secondary confirmative test. Similarly, increased (or incorrect) diagnosis of TB may compromise programme effectiveness by increasing the numbers of individuals requiring anti-tuberculosis treatment, unless it is carefully planned. For the future, 1) assays with greater sensitivity for TB detection are needed; 2) rapid diagnostics for paediatric TB are important, and there is a need for carefully designed studies, including those involving HIV-positive children; 3) more clinical data need to be obtained from longitudinal studies, especially related to the influence of rapid diagnostics on disease outcome; and 4) point-of-care tests using untreated sputum, blood or urine and little or no equipment would be of immeasurable benefit. Although great progress has been made, we are not there yet.

Rapid and inexpensive drug susceptibility testing for Mycobacterium tuberculosis by a nitrate reductase assay (NRA) from western India

Increase in multi-drug resistant (MDR) Mycobacterium tuberculosis (MTB) strains is a worrisome trend seen during recent years. Rapid detection of MDR strains is very important to restrict their spread in the population. Gold standard methods for drug susceptibility testing (DST) of MTB are either costly or very slow. Nitrate reductase assay (NRA) is one of the methods for rapid detection of resistance. This technique is based on the capacity of M. tuberculosis to reduce nitrate to nitrite. The WHO recommends that the NRA be used as direct test on smear-positive sputum specimen or as an indirect test on Mycobacterium tuberculosis isolates grown from conventional solid cultures. We evaluated the performance of NRA as rapid, reliable & inexpensive method for drug-susceptibility testing of Mycobacterium tuberculosis against first line antitubercular drugs, Rifampicin (RIF) and Isoniazid (INH).

Evaluation of a Novel Biphasic Culture Medium for Recovery of Mycobacteria: A Multi-Center Study

BackgroundMycobacterial culture and identification provide a definitive diagnosis of TB. Culture on Löwenstein-Jensen (L-J) medium is invariably delayed because of the slow growth of M. tuberculosis on L-J slants. Automated liquid culture systems are expensive. A low-cost culturing medium capable of rapidly indicating the presence of mycobacteria is needed. The aim of this study was to develop and evaluate a novel biphasic culture medium for the recovery of mycobacteria from clinical sputum specimens from suspected pulmonary tuberculosis patients.Methods and FindingsThe biphasic medium consisted of 7 ml units of L-J slant medium, 3 ml units of liquid culture medium, growth indicator and a mixture of antimicrobial agents. The decontamination sediments of sputum specimens were incubated in the biphasic culture medium at 37°C. Mycobacterial growth was determined based on the appearance of red granule sediments and the examination using acid-fast bacilli (AFB). The clinical sputum specimens were cultured in the biphasic medium, on L-J slants and in the Bactec MGIT 960 culture system. Among smear-positive specimens, the mycobacteria recovery rate of the biphasic medium was higher than that of the L-J slants (P<0.001) and similar to that of MGIT 960 (P>0.05). Among smear-negative specimens, the mycobacterial recovery rate of the biphasic medium was higher than that of L-J slants (P<0.001) and lower than that of MGIT 960 (P<0.05). The median times to detection of mycobacteria were 14 days, 20 days and 30 days for cultures grown in MGIT, in biphasic medium, on L-J slants for smear negative specimens, respectively (P<0.001).ConclusionsThe biphasic culture medium developed in this study is low-cost and suitable for mycobacterial recovery. It does not require any expensive detection instrumentation, decreases the time required for detection of M. tuberculosis complex, and increases the detection rate of M. tuberculosis complex.

Real-Time Molecular Epidemiology of Tuberculosis by Direct Genotyping of Smear-Positive Clinical Specimens

We applied MIRU-VNTR (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing) to directly analyze the bacilli present in 61 stain-positive specimens from tuberculosis patients. A complete MIRU type (24 loci) was obtained for all but one (no amplification in one locus) of the specimens (98.4%), and the allelic values fully correlated with those obtained from the corresponding cultures. Our study is the first to demonstrate that real-time genotyping of Mycobacterium tuberculosis can be achieved, fully transforming the way in which molecular epidemiology techniques can be integrated into control programs.

Rapid and effective diagnosis of tuberculosis and rifampicin resistance with Xpert MTB/RIF assay: A meta-analysis

Xpert MTB/RIF (Cepheid) assay has been introduced for the diagnosis of tuberculosis (TB) and RIF-resistance. The meta-analysis was used to establish the overall accuracy of Xpert MTB/RIF assay for diagnosing TB and RIF-resistance. Based on comprehensive searches of the Pubmed and Embase, we identified outcome data from all articles estimating diagnostic accuracy with Xpert MTB/RIF assay. A summary estimation for sensitivity, specificity, diagnostic odds ratios (DOR) and the area under the summary ROC curve (AUC) was calculated by using the bivariate random-effects approach. The meta-analysis included 18 studies (10,224 suspected specimens). The summary estimate was 90.4% (95%CI 89.2%-91.4%) for sensitivity, 98.4% (95%CI 98.0%-98.7%) for specificity and 328.3/0.9822 for DOR/AUC in pulmonary tuberculosis (PTB). The sensitivity, specificity and DOR/AUC of detecting RIF-resistance were 94.1%, 97.0% and 177.8/0.9832, respectively. For extrapulmonary tuberculosis, the overall pooled sensitivity was 80.4% and specificity was 86.1%. The findings in subgroup analysis were as follows: the accuracy of Xpert MTB/RIF assay is higher in smear-positive specimens and the sensitivity of diagnosing PTB in adults was higher than that in children (90.8% versus 74.3%). TB and RIF-resistance can be rapidly and effectively diagnosed with Xpert MTB/RIF assay.

Rapid Direct Testing of Susceptibility of Mycobacterium tuberculosis to Isoniazid and Rifampin on Nutrient and Blood Agar in Resource-Starved Settings

In this study, we evaluated the performance of blood agar (by macroscopic growth) and nutrient agar (by a microcolony detection method) for drug susceptibility testing of Mycobacterium tuberculosis against rifampin (RIF) and isoniazid (INH), using 67 smear-positive sputum specimens. The direct proportion method on Lowenstein-Jensen (LJ) medium was used as the "gold standard." Compared with LJ medium, results for both media were in 100% agreement for RIF, while for INH the agreement levels for blood agar and nutrient agar were 98% and 95%, respectively. Within 2 weeks, 100% of specimens yielded results on blood agar, while 96.8% of specimens yielded results on nutrient agar. Our study showed that blood agar and nutrient agar can be used as alternative media for direct susceptibility testing of RIF and INH, especially in resource-poor settings.

First insight into the genetic diversity of Mycobacterium tuberculosis strains from patients in Duhok, Iraq

BackgroundThe aim of this study was to evaluate the genotypic diversity of Mycobacterium tuberculosis strains using IS6110 RFLP, spoligotyping and MIRU-VNTR typing. Materials and methodsBetween June 2008 and June 2009, all smear positive pulmonary specimens were collected prospectively at the national tuberculosis program (NTP) center of Duhok province. The specimens were processed for culture by modified Petroff’s method and were inoculated into two tubes of Löwenstein-Jensen (LJ) media. The isolates were identified as M. tuberculosis by using biochemical tests and growth rate. Molecular fingerprinting of all M. tuberculosis strains was performed by IS6110 RFLP, spoligotyping and MIRU-VNTR. ResultsM. tuberculosis strains were isolated from 53 Iraqi patients with pulmonary TB. Spoligotyping of M. tuberculosis isolates showed T family (30%) as the predominant genotype. By using the three molecular techniques, there were four spoligotyping clusters of strains (“3540 & 3516”, “3565 & 3563”, “3605 & 3618” and “865, 877 & 13811”). Complete concordance with RFLP was observed in one cluster of spoligotyping, but no concordance with MIRU-VNTR profile (234426153433 and 236424183433). ConclusionsMolecular fingerprinting methods are vital for differentiating a reactivation of latent infection from a recent transmission; however, it should be coupled with clinical epidemiological investigation. The low clustering rate in this study suggests that either reactivation of latent infections may be the main driving force for the endemic situation of the disease in Duhok, or it may indicate that a big circle of TB transmission is missed in the community, which means effective control measures have not been achieved yet in Duhok.

Rapid molecular testing for multi-resistant tuberculosis in Mongolia: A diagnostic accuracy study

ObjectiveThe aim of this study was to assess the performance of a molecular line probe assay, GenoType® MTBDRplus, for rapid detection of rifampicin and isoniazid resistance in the Mongolian situation. The sensitivity and specificity of GenoType® MTBDRplus to detect rifampicin and isoniazid resistance-associated mutations in culture specimens and directly in smear-positive clinical specimens was examined. Method218 MDR-TB subjects aged between 14 and 75years old from eight districts in Ulaanbaatar city (between July 2009 and May 2010) were included in this study .The GenoType Mycobacterium tuberculosis drug resistance first line (MTBDR plus) assay (Hain Life-science, Nehren, Germany) was tested on 109 clinical isolates and directly on 41 sputum specimens for the ability to detect the resistances. Results were compared with conventional culture and drug susceptibility testing on solid medium. ResultsThe high correlation of the results from GenoType® MTBDRplus and conventional drug susceptibility testing was obtained from this study. The results clearly showed a high performance of GenoType® MTBDRplus with almost 100% accuracy for all the important indicators, such as sensitivity, specificity, positive and negative predictive values and detection of rifampicin resistance. Discrepancies were obtained in comparison with DNA sequencing results. ConclusionsThe Genotype® MTBDRplus assay was demonstrated as a rapid, reliable and highly accurate tool for early detection of MDR-TB through examining smear positive cases.

Use of a Molecular Diagnostic Test in AFB Smear Positive Tuberculosis Suspects Greatly Reduces Time to Detection of Multidrug Resistant Tuberculosis

BackgroundThe WHO has recommended the implementation of rapid diagnostic tests to detect and help combat M/XDR tuberculosis (TB). There are limited data on the performance and impact of these tests in field settings.MethodsThe performance of the commercially available Genotype MTBDRplus molecular assay was compared to conventional methods including AFB smear, culture and drug susceptibility testing (DST) using both an absolute concentration method on Löwenstein-Jensen media and broth-based method using the MGIT 960 system. Sputum specimens were obtained from TB suspects in the country of Georgia who received care through the National TB Program.ResultsAmong 500 AFB smear-positive sputum specimens, 458 (91.6%) had both a positive sputum culture for Mycobacterium tuberculosis and a valid MTBDRplus assay result. The MTBDRplus assay detected isoniazid (INH) resistance directly from the sputum specimen in 159 (89.8%) of 177 specimens and MDR-TB in 109 (95.6%) of 114 specimens compared to conventional methods. There was high agreement between the MTBDRplus assay and conventional DST results in detecting MDR-TB (kappa = 0.95, p<0.01). The most prevalent INH resistance mutation was S315T (78%) in the katG codon and the most common rifampicin resistance mutation was S531L (68%) in the rpoB codon. Among 13 specimens from TB suspects with negative sputum cultures, 7 had a positive MTBDRplus assay (3 with MDR-TB). The time to detection of MDR-TB was significantly less using the MTBDRplus assay (4.2 days) compared to the use of standard phenotypic tests (67.3 days with solid media and 21.6 days with broth-based media).ConclusionsCompared to conventional methods, the MTBDRplus assay had high accuracy and significantly reduced time to detection of MDR-TB in an area with high MDR-TB prevalence. The use of rapid molecular diagnostic tests for TB and drug resistance should increase the proportion of patients promptly placed on appropriate therapy.

Real-time PCR and Amplified MTD® for rapid detection of Mycobacterium tuberculosis in pulmonary specimens

Patients with suspected pulmonary tuberculosis (PTB) in the First Affiliated Hospital, College of Medicine, Zhejiang University. To evaluate the 'Care TB®' real-time polymerase chain reaction (PCR) assay in comparison with the Amplified MTD® Test (AMTD) for the rapid diagnosis of TB. Self-expectorated sputum was collected for direct smear microscopy, culture, real-time PCR and AMTD assay. Performance of the 'Care TB' real-time PCR and AMTD assay were compared using a combination of culture and clinical diagnosis as a reference standard. Of the 178 sputum specimens, 83 were culture-positive; of these, 74 were Mycobacterium tuberculosis complex and 9 strains were non-tuberculous mycobacteria. The overall sensitivities and specificities were respectively 91.6% and 100% for real-time PCR, and 95.2% and 97.9% for AMTD. In the smear-positive specimens, the sensitivities and specificities were respectively 97% and 100% for real-time PCR, and 98.5% and 100% for AMTD. In the smear-negative specimens, the sensitivities and specificities were 70.6% and 100% for real-time PCR, and 82.4% and 97.7% for AMTD. Both real-time PCR and AMTD are rapid and specific tests for detecting M. tuberculosis complex; however, 'Care TB' real-time PCR is more convenient and economical.

Rapid molecular detection of tuberculosis and rifampicin drug resistance: retrospective analysis of a national UK molecular service over the last decade

BackgroundFast and reliable detection of Mycobacterium tuberculosis complex (MTBC) and drug resistance is crucial in establishing effective treatment and enforcing timely public health measures.MethodsThe authors analysed the performance of a...

Utility of GenoType MTBDRplus assay in rapid diagnosis of multidrug resistant tuberculosis at a tertiary care centre in India

Purpose: Molecular methods which allow rapid detection of tuberculosis as well as drug resistance directly from clinical samples have become the most popular diagnostic methodology with the emergence of multidrug resistant tuberculosis. The aim of the present study was to evaluate the performance of a line probe assay, GenoType MTBDRplus for the rapid detection of Mycobacterium tuberculosis and mutations causing rifampicin and INH resistance directly in smear positive pulmonary specimens and also in M. tuberculosis isolates grown from various clinical specimens. Materials and Methods: The MTBDRplus assay was done directly on 37 smear positive pulmonary specimens and also on 69 M. tuberculosis isolates obtained by rapid automated culture using Bact/Alert 3D. The results were compared with phenotypic drug susceptibility testing (1% proportion method) using Bact/Alert 3D. Results: The sensitivity and specificity for detection of resistance to rifampicin was 100% and 97.3%, and to INH was 91.9% and 98.4%, respectively, in comparison with the phenotypic drug susceptibility testing. Conclusion: MTBDRplus assay had good sensitivity and specificity with turn around time of less than 48 hours. It may be a useful tool for rapid detection of multidrug resistant tuberculosis at a tertiary care centre.

Novel Real-Time Simultaneous Amplification and Testing Method To Accurately and Rapidly Detect Mycobacterium tuberculosis Complex

The aim of this study was to establish and evaluate a simultaneous amplification and testing method for detection of the Mycobacterium tuberculosis complex (SAT-TB assay) in clinical specimens by using isothermal RNA amplification and real-time fluorescence detection. In the SAT-TB assay, a 170-bp M. tuberculosis 16S rRNA fragment is reverse transcribed to DNA by use of Moloney murine leukemia virus (M-MLV) reverse transcriptase, using specific primers incorporating the T7 promoter sequence, and undergoes successive cycles of amplification using T7 RNA polymerase. Using a real-time PCR instrument, hybridization of an internal 6-carboxyfluorescein-4-[4-(dimethylamino)phenylazo] benzoic acid N-succinimidyl ester (FAM-DABCYL)-labeled fluorescent probe can be used to detect RNA amplification. The SAT-TB assay takes less than 1.5 h to perform, and the sensitivity of the assay for detection of M. tuberculosis H37Rv is 100 CFU/ml. The TB probe has no cross-reactivity with nontuberculous mycobacteria or other common respiratory tract pathogens. For 253 pulmonary tuberculosis (PTB) specimens and 134 non-TB specimens, the SAT-TB results correlated with 95.6% (370/387 specimens) of the Bactec MGIT 960 culture assay results. The sensitivity, specificity, and positive and negative predictive values of the SAT-TB test for the diagnosis of PTB were 67.6%, 100%, 100%, and 62.0%, respectively, compared to 61.7%, 100%, 100%, and 58.0% for Bactec MGIT 960 culture. For PTB diagnosis, the sensitivities of the SAT-TB and Bactec MGIT 960 culture methods were 97.6% and 95.9%, respectively, for smear-positive specimens and 39.2% and 30.2%, respectively, for smear-negative specimens. In conclusion, the SAT-TB assay is a novel, simple test with a high specificity which may enhance the detection rate of TB. It is therefore a promising tool for rapid diagnosis of M. tuberculosis infection in clinical microbiology laboratories.

Direct Drug Susceptibility Testing of Mycobacterium tuberculosis for Rapid Detection of Multidrug Resistance Using the Bactec MGIT 960 System: a Multicenter Study

Conventional indirect drug susceptibility testing of Mycobacterium tuberculosis with liquid medium is well established and offers time-saving and reliable results. This multicenter study was carried out to evaluate if drug susceptibility testing (DST) can be successfully carried out directly from processed smear-positive specimens (direct DST) and if this approach could offer substantial time savings. Sputum specimens were digested, decontaminated, and concentrated by the laboratory routine procedure and were inoculated in Bactec MGIT 960 as well as Lowenstein-Jensen (LJ) medium for primary isolation. All the processed specimens which were acid-fast bacterium (AFB) smear positive were used for setting up direct DST for isoniazid (INH) and rifampin (RIF). After the antimicrobial mixture of polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin (PANTA) was added, the tubes were entered in the MGIT 960 instrument using the 21-day protocol (Bactec 960 pyrazinamide [PZA] protocol). Results obtained by direct DST were compared with those obtained by indirect DST to establish accuracy and time savings by this approach. Of a total of 360 AFB smear-positive sputum specimens set up for direct DST at four sites in three different countries, 307 (85%) specimens yielded reportable results. Average reporting time for direct DST was 11 days (range, 10 to 12 days). The average time savings by direct DST compared to indirect DST, which included time to isolate a culture and perform DST, was 8 days (range, 6 to 9 days). When results of direct DST were compared with those of indirect DST, there was 95.1% concordance with INH and 96.1% with rifampin. These findings indicate that direct DST with the Bactec MGIT 960 system offers further time savings and is a quick method to reliably detect multidrug resistance (MDR) cases.

Evaluation of the GeneXpert MTB/RIF Assay for Rapid Diagnosis of Tuberculosis and Detection of Rifampin Resistance in Pulmonary and Extrapulmonary Specimens

Mycobacterium tuberculosis remains one of the most significant causes of death from an infectious agent. The rapid diagnosis of tuberculosis and detection of rifampin (RIF) resistance are essential for early disease management. The GeneXpert MTB/RIF assay is a novel integrated diagnostic device for the diagnosis of tuberculosis and rapid detection of RIF resistance in clinical specimens. We determined the performance of the MTB/RIF assay for rapid diagnosis of tuberculosis and detection of rifampin resistance in smear-positive and smear-negative pulmonary and extrapulmonary specimens obtained from possible tuberculosis patients. Two hundred fifty-three pulmonary and 176 extrapulmonary specimens obtained from 429 patients were included in the study. One hundred ten (89 culture positive and 21 culture negative for M. tuberculosis) of the 429 patients were considered to have tuberculosis. In pulmonary specimens, sensitivities were 100% (27/27) and 68.6% (24/35) for smear-positive and smear-negative specimens, respectively. It had a lower sensitivity with extrapulmonary specimens: 100% for smear-positive specimens (4/4) and 47.7% for smear-negative specimens (21/44). The test accurately detected the absence of tuberculosis in all 319 patients without tuberculosis studied. The MTB/RIF assay also detected 1 RIF-resistant specimen and 88 RIF-susceptible specimens, and the results were confirmed by drug susceptibility testing. We concluded that the MTB/RIF test is a simple method, and routine staff with minimal training can use the system. The test appeared to be as sensitive as culture with smear-positive specimens but less sensitive with smear-negative pulmonary and extrapulmonary specimens that include low numbers of bacilli.